Recombinant Neurospora crassa NAD (P)H-hydrate epimerase (NCU08070)

What is Neurospora crassa NAD(P)H-hydrate epimerase and what is its primary function?

NAD(P)H-hydrate epimerase (encoded by the NCU08070 gene in Neurospora crassa) is an enzyme that catalyzes the interconversion between R and S epimers of hydrated NAD(P)H (NAD(P)HX). This enzyme is part of a two-enzyme NAD(P)HX repair system that restores damaged/hydrated forms of NAD(P)H back to their active forms. The repair system involves the epimerase (which converts the R form to the S form of NAD(P)HX) and a dehydratase that specifically acts on the S-form to regenerate NAD(P)H . This repair mechanism is critical because NAD(P)HX can inhibit various dehydrogenases and accumulation of these damaged forms can have detrimental effects on cellular metabolism .

How is the NCU08070 gene designated in databases and what are its key features?

The NCU08070 gene in Neurospora crassa is also designated as NNRE_NEUCR in some databases. It encodes the NAD(P)H-hydrate epimerase protein with UniProt accession number Q7SGL3. The protein is found in Neurospora crassa strain ATCC 24698/74-OR23-1A/CBS 708.71/DSM 1257/FGSC 987 (taxonomy ID: 367110) . The protein participates in at least 386 full-length protein ortholog groups according to the InParanoiDB database, indicating its high conservation across species .

What is the structural basis for NAD(P)H-hydrate epimerase function?

The functional mechanism of NAD(P)H-hydrate epimerase involves specific active site residues that facilitate the epimer interconversion. A conserved lysine residue (K192 in E. coli YjeF) is crucial for epimerase activity . Structural studies have shown that mutation of this residue can reduce in vitro epimerase activity by ≥95%. Crystal structures of related epimerases with bound substrates have revealed the structural basis for substrate recognition and selectivity. The enzyme typically contains a conserved domain architecture that can be visualized through domain ortholog group analysis .

What methods are used to express and purify recombinant NCU08070 protein?

Recombinant Neurospora crassa NAD(P)H-hydrate epimerase can be produced using various expression systems, with baculovirus expression systems being one common approach . The methodological process typically involves:

• Cloning the NCU08070 gene into an appropriate expression vector

• Transforming/transfecting the construct into the expression host (insect cells for baculovirus systems)

• Inducing protein expression under optimized conditions

• Cell lysis and initial clarification by centrifugation

• Purification using affinity chromatography (often His-tag based)

• Further purification steps such as ion exchange and size exclusion chromatography

• Quality assessment using SDS-PAGE, Western blotting, and enzymatic activity assays

The final purified protein can then be used for various biochemical, structural, and functional studies .

How is the enzymatic activity of NAD(P)H-hydrate epimerase measured experimentally?

The enzymatic activity of NAD(P)H-hydrate epimerase is typically assessed through coupled enzyme assays that monitor the restoration of NAD(P)H. Methodological approaches include:

• Generation of NAD(P)HX substrates by controlled heat and acid treatment of NAD(P)H

• Incubation of the enzyme with the generated R/S epimer mixtures

• Measurement of NAD(P)H regeneration after addition of the dehydratase (which acts only on the S-epimer)

• Detection through spectrophotometric monitoring at 340 nm (absorption maximum of NAD(P)H)

• Calculation of kinetic parameters (kcat, KM, kcat/KM)

For the TM0006 protein from Thermotoga maritima (related to NCU08070), studies showed values of kcat/KM in the 104 M-1s-1 range for substrates like L-Ala-L-Phe and L-Ala-L-His, confirming efficient epimerase activity .

Table adapted from kinetic data of related epimerase enzymes

What genetic approaches can be used to study the function of NCU08070 in Neurospora crassa?

Several genetic and molecular biology techniques can be applied to investigate NCU08070 function:

• Gene deletion/knockout: Using homologous recombination strategies similar to those described for nox-1 and nox-2 deletion in N. crassa, researchers can generate NCU08070 deletion strains

• Site-directed mutagenesis: Introduction of specific mutations to study structure-function relationships

• GFP-tagging for localization studies: Similar to approaches used for PALA protein in N. crassa

• RNA interference (RNAi): For conditional knockdown when complete deletion is not desirable

• Transcriptome analysis: RNA-Seq to examine expression changes in response to environmental conditions or after gene manipulation

• Complementation studies: To verify gene function and for cross-species functional analysis

These approaches can reveal the biological roles of NCU08070 in vivo and its potential connections to other metabolic pathways .

What evidence exists for NAD(P)H-hydrate epimerase having additional functions beyond NAD(P)HX repair?

Several lines of evidence suggest that NAD(P)H-hydrate epimerase may have functions beyond metabolite repair:

• Bioinformatic analysis reveals that in plants, the epimerase is fused to a vitamin B6 salvage enzyme

• Chromosomal clustering analysis shows that in bacteria, epimerase genes often cluster with genes related to vitamin B6 metabolism, including PLP-dependent enzymes like alanine racemase and glutamate decarboxylase

• Coexpression data from yeast and Arabidopsis demonstrate that epimerase genes correlate strongly with PLP-dependent enzymes and genes involved in amino acid metabolism

• Mutational studies in E. coli show that cells with a K192A mutation (which disrupts epimerase activity but maintains normal NAD(P)HX repair in vivo through the dehydratase) exhibit metabolome changes that exceed those in cells completely lacking the yjeF gene, particularly in amino acid pathways

• These mutant cells also showed reduced levels of free pyridoxal 5'-phosphate, providing direct evidence of a connection to vitamin B6 metabolism

These findings collectively suggest that NAD(P)H-hydrate epimerase has evolved to perform additional functions, potentially as a moonlighting protein involved in vitamin B6-related processes .

How does the NAD(P)HX repair system in Neurospora crassa compare to other species, and what are the evolutionary implications?

The NAD(P)HX repair system shows remarkable conservation across all domains of life, indicating its fundamental importance in metabolism. Comparative analysis reveals:

• Organization differences: In E. coli, the epimerase and dehydratase are fused as a single protein (YjeF), while they exist as separate proteins in Neurospora crassa and mammals

• Phylogenetic distribution: Analysis of over 1,600 prokaryotic genomes found that 1,248 contained both enzymes, with 97% having them fused together, while 168 genomes had only the dehydratase

• Substrate specificity variation: Some related enzymes, like those from Thermotoga maritima, show different substrate specificities (e.g., for dipeptides with alanine in the first position and aromatic amino acids in the second), suggesting functional divergence

• Extracellular presence: In mammals, the epimerase (NAXE) is found in extracellular compartments (cerebrospinal fluid, urine, plasma), while the dehydratase is not, suggesting additional functions

• Clinical significance: Deficiency of these enzymes in humans causes fatal neurometabolic disorders, highlighting their critical role in human health

The evolutionary pattern suggests that while the core repair function is preserved, the epimerase component has acquired additional roles in different lineages, potentially contributing to species-specific metabolic adaptations .

What is the relationship between NAD(P)H-hydrate epimerase and stress response in Neurospora crassa?

The relationship between NAD(P)H-hydrate epimerase and stress response mechanisms represents an important research area:

• Metabolic stress: Under conditions of metabolic stress (heat, acidity), the nonenzymatic conversion of NAD(P)H to NAD(P)HX increases, potentially elevating the demand for epimerase activity

• Inflammatory stress: In humans, NAD(P)HX epimerase deficiency causes fatal neurometabolic disorder with decompensations precipitated specifically by inflammatory stress, suggesting a connection to inflammatory response mechanisms

• Oxidative stress: Research in related systems suggests connections to oxidative stress response pathways. In N. crassa, NOX-1 (NADPH oxidase) is involved in ROS production and sexual development, potentially intersecting with pathways requiring robust NAD(P)H homeostasis

• Nitric oxide response: Studies on nitric oxide scavenging in N. crassa reveal transcriptomic changes that could potentially involve NAD(P)H-dependent processes

• Calcium signaling: Investigations in N. crassa show connections between stress responses, calcium signaling, and NAD(P)H-dependent processes

These connections suggest that NCU08070 may play roles in maintaining metabolic integrity during various stress conditions, potentially through both its primary repair function and possible moonlighting activities .

What is known about the interaction between Neurospora crassa NAD(P)H-hydrate epimerase and other metabolic pathways?

The NAD(P)H-hydrate epimerase in N. crassa potentially interacts with several metabolic networks:

• NAD(P)H-dependent dehydrogenases: These enzymes can be inhibited by NAD(P)HX, making the epimerase crucial for maintaining their function

• Vitamin B6 metabolism: Bioinformatic and experimental evidence points to connections with pyridoxal 5'-phosphate (PLP) metabolism and PLP-dependent enzymes

• Amino acid metabolism: Mutations affecting epimerase function in related systems show perturbations in amino acid metabolic pathways

• Redox homeostasis: As part of NAD(P)H metabolism, the epimerase indirectly influences cellular redox state and potentially ROS-related signaling

• Energy metabolism: Through its effects on NAD(P)H availability, the epimerase may impact energy-generating pathways

• Bacterial interaction pathways: Recent studies show N. crassa interacts with Pseudomonas syringae, with potential involvement of NAD(P)H-dependent processes

These interconnections position NAD(P)H-hydrate epimerase as a node in a complex metabolic network rather than an isolated repair enzyme .

How can insights from NCU08070 be applied to understand human NAXE-related disorders?

Neurospora crassa NAD(P)H-hydrate epimerase (NCU08070) research has significant translational potential for understanding human NAXE-related disorders:

• Mechanistic insights: Detailed biochemical and structural studies of the fungal enzyme can reveal fundamental mechanisms of NAD(P)HX repair applicable to the human system

• Pathogenic mutations: Modeling human disease-causing mutations in the N. crassa enzyme can help understand their biochemical consequences in a tractable experimental system

• Metabolic signatures: The metabolomic changes observed in experimental systems lacking functional epimerase activity can guide biomarker discovery for human NAXE disorders, such as:

  • Depletion of NAD+ and its catabolites (ADPR, MNA, 2PY)

  • Signs of mitochondrial dysfunction

  • Altered lipidomics profiles

• Therapeutic strategies: Understanding the success of niacin supplementation in both model systems and human cases provides a foundation for developing improved treatments

• Stress response mechanisms: Elucidating the specific connections between inflammatory stress and NAD(P)HX accumulation could identify new therapeutic targets

This research exemplifies how fungal model systems can provide crucial insights into human metabolic disorders .

What methodological approaches can be used to explore the potential moonlighting functions of NAD(P)H-hydrate epimerase?

Investigating the proposed moonlighting functions of NAD(P)H-hydrate epimerase requires multifaceted approaches:

• Targeted mutagenesis strategies:

  • Creating separation-of-function mutations (like K192A) that specifically disrupt epimerase activity while preserving protein structure

  • Generating chimeric proteins that swap domains between species with different functional profiles

• Protein interaction studies:

  • Immunoprecipitation followed by mass spectrometry

  • Yeast two-hybrid screening

  • Proximity labeling approaches (BioID, APEX)

  • Analysis of protein-protein interaction networks

• Metabolomic approaches:

  • Untargeted metabolomics to identify broader metabolic impacts

  • Targeted analysis of vitamin B6-related metabolites

  • Stable isotope tracing to track metabolic fluxes

  • Comparative metabolomics of wild-type, null mutant, and separation-of-function mutant strains

• Transcriptomic analysis:

  • RNA-Seq under various conditions (like those used for nitric oxide response studies in N. crassa)

  • Correlation analysis between epimerase expression and vitamin B6-related genes

  • Response to vitamin B6 supplementation or depletion

• Structural biology approaches:

  • Co-crystallization with potential alternative substrates

  • Molecular docking simulations

  • Nuclear magnetic resonance (NMR) studies of protein-ligand interactions

These approaches would help delineate the full functional repertoire of this fascinating enzyme .

How do environmental factors influence the expression and activity of NCU08070 in Neurospora crassa?

Environmental regulation of NCU08070 represents an important research direction:

Understanding these regulatory patterns would provide insights into when and how the NAD(P)HX repair system is mobilized in response to cellular needs .